Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

pone-0013470-g001: Overview of approach.Human lung cancer cell lines (Suppl. Table S1) were cultured in the presence or absence of tyrosine kinase inhibitors erlotinib or dasatinib. Cell proteins were extracted and analyzed by rosette and far-Western blotting using an array of SH2 domain probes (Suppl. Table S3). Bioinformatic analysis of quantified data was used for classification and biomarker screening.

Mentions:
We selected a group of 22 non-small cell lung cancer cell lines with known EGFR and K-RAS mutation status and known sensitivity to the EGFR TKI erlotinib (Suppl. Table S1). The overall strategy for our studies is shown in Fig. 1. Cell lysates were prepared from actively growing cells cultured in serum-supplemented medium. Two approaches to generate SH2 profiles were used, reverse-phase protein array and far-Western blotting [28]. In the first method, multiple protein samples (cell lysates) are spotted in arrays in register with the wells of a 96-well chamber apparatus. Each well is then filled with a solution containing a different GST-SH2 domain probe, and after incubation and washing, the bound probe is quantified for each spot. The amount of binding depends on the number and affinity of tyrosine phosphorylated protein sites in the sample. With this approach, which we term the “rosette” assay, it is possible to profile the total level of binding for virtually all SH2 domains in the genome (94 SH2 domain probes and one PTB domain representing 90 distinct proteins) using minimal amounts of protein sample.

pone-0013470-g001: Overview of approach.Human lung cancer cell lines (Suppl. Table S1) were cultured in the presence or absence of tyrosine kinase inhibitors erlotinib or dasatinib. Cell proteins were extracted and analyzed by rosette and far-Western blotting using an array of SH2 domain probes (Suppl. Table S3). Bioinformatic analysis of quantified data was used for classification and biomarker screening.

Mentions:
We selected a group of 22 non-small cell lung cancer cell lines with known EGFR and K-RAS mutation status and known sensitivity to the EGFR TKI erlotinib (Suppl. Table S1). The overall strategy for our studies is shown in Fig. 1. Cell lysates were prepared from actively growing cells cultured in serum-supplemented medium. Two approaches to generate SH2 profiles were used, reverse-phase protein array and far-Western blotting [28]. In the first method, multiple protein samples (cell lysates) are spotted in arrays in register with the wells of a 96-well chamber apparatus. Each well is then filled with a solution containing a different GST-SH2 domain probe, and after incubation and washing, the bound probe is quantified for each spot. The amount of binding depends on the number and affinity of tyrosine phosphorylated protein sites in the sample. With this approach, which we term the “rosette” assay, it is possible to profile the total level of binding for virtually all SH2 domains in the genome (94 SH2 domain probes and one PTB domain representing 90 distinct proteins) using minimal amounts of protein sample.

Affiliation:
Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.